The invention relates generally to automated methods and devices for iterative staining of biological samples for imaging applications.
Biological samples are used for analytic and diagnostic purposes, such as diagnosing diseased tissue at a molecular level. A biological sample, such as tissue micro arrays (TMA), on which tissue samples are fixed are typically stained with a morphological stain or biomarker. The stained TMA are then analyzed manually with a microscope, or an image may be taken of the TMA for subsequent analysis or comparison. After the first stain is applied and imaged, one or more serial or successive stains or biomarkers may be applied and the TMA may be analyzed again. The two or more serial images may then be compared. A single staining cycle may comprise the steps of flowing a stain (antibody) over the tissue, incubating the stain for an appropriate time, rinsing away the stain to reduce background fluorescence, imaging the slide, and bleaching away the stain. As part of the multiplexing technique developed for fluorescent imaging, sequential staining, rinsing, and destaining cycles are required. The staining cycle may be then repeated for multiple stains. For multiplexed applications, the TMA needs to be stained with multiple molecular probes to investigate protein expression or spatial distribution quantitatively or qualitatively.
The staining process is typically performed using time-consuming manual techniques that are susceptible to error. Thus, the total operation time is the sum of each of these steps multiplied by the total number of applied stains. Currently, time for each step is determined based on the amount of fluids required for each step. The time for each step is fixed independent of which antibody (stain) is being applied. Thus a very weak stain will be subjected to the same bleaching time as a much stronger stain, even though a shorter bleaching time may suffice. The reagents used in the staining process are often expensive and have limited shelf life thereby requiring special handling techniques
Therefore, it is desirable to provide a method for optimizing one or more steps in a staining cycle. It is also desirable to automate the process and reduce manual intervention to make the process time efficient and reliable.